1. Field of the Invention
The invention relates to transiently binding a drag-tag to a molecule during a separation modality.
2. Description of Related Art
The ultimate success of a DNA sequencing methodology is determined by both the resolving power of the technique itself and its sensitivity to small differences in migrational velocity over a broad range of sequencing fragment lengths. For direct comparison of sequencing efficiency between various sequencing methodologies, it is often convenient to cite the length of read (“LOR”)/unit time, usually in the number of called bases/day. Current capillary gel electrophoresis (“CGE”)-based separation approaches have a LOR of approximately 500 to 600 bases with run times on the order of 2.5 hours. This would translate into approximately 5,000 bases/day for a single capillary instrument. For a 96 capillary array, this value approaches 500,000 bases/day, clearly indicating the advantage of massively parallel separations. This LOR is used as an indication of a technique's sequencing capacity. In CGE-based techniques, the major obstacle to longer sequencing read lengths is diffusion band broadening, a result of the long run times required to avoid biased reptation.
End labeled free solution electrophoresis (ELFSE) is a DNA separation modality capable of breaking the charge-to-friction ratio of DNA in a gel- or polymer-free context. At the foundation of this particular technique lies the notion that by appending a drag inducing entity (i.e. a “drag-tag”), predominantly one with little or no charge, the charge-to-friction symmetry of the DNA target can be broken, and the resulting DNA fragment can be separated. Since DNA acts as a free-draining coil, the electrophoretic mobility of DNA can roughly be described by q/f, or its charge-to-friction ratio, where q is the net charge on the molecule and f is the molecular friction coefficient. By covalently attaching an uncharged drag-tag to the DNA target, the friction induced by the pendant drag-tag moiety decreases the electrophoretic mobility through an increase in the hydrodynamic friction of the resulting complex. The drag-tag/DNA complex thereby possesses a charge equivalent to the DNA itself, while the combined hydrodynamic friction of the drag-tag/DNA complex is increased by the hydrodynamic friction of the entire complex, including the drag-tag.
Since the inception of ELFSE methods, a number of obstacles remain to be overcome for the successful application of this methodology. Since ELFSE-based approaches separate through inherently different physical mechanisms, biased reptation is not a concern, and substantially higher electric field strengths (and the short run times that would result) are achievable, representing an opportunity for improved separation performance. Unfortunately, there are entirely different limitations with ELFSE. The lack of sufficiently large, monodispersed polymeric drag-tags currently limits the achievable LOR for ELFSE based approaches to around 125 bases.